A variety of proteins exist in biological samples, and methods such as ELISA (Enzyme-Linked ImmunoSorbent Assay) are known as methods for detecting and quantifying specific proteins.
ELISA is a method for quantitatively detecting a specific protein such as an antigen contained in a sample, by using an enzyme-labeled antibody and utilizing an antigen-antibody reaction, and is one technique that is widely used in immunological tests and the like. Known forms of ELISA include the direct adsorption method, the sandwich method and the competitive method.
For example, a primary antibody for a target material (antigen) adsorbed to the surface of a solid phase is bound via an antigen-antibody reaction. The unreacted primary antibody is washed away, and an enzyme-labeled secondary antibody is then added and bound via a second antigen-antibody reaction. The unreacted labeled secondary antibody is then washed away, and when a chromogenic substrate is added, a color reaction occurs in proportion to the amount of the antigen. The absorbance of the thus generated colored material is measured using an absorbance meter or the like, and the amount of the antigen can be quantified by using a calibration curve prepared using standard samples of known concentration.
However, in this type of method, a labeled secondary antibody is required which binds specifically to the primary antibody that binds specifically to the target material (antigen), and when a plurality of types of target materials (antigens) are to be detected, a series of labeled secondary antibodies must be prepared to bind specifically to each of the plurality of primary antibodies, meaning the method suffers from poor versatility.
Alkaline phosphatases are well known as enzymes for protein detection, and among such alkaline phosphatases, CIAP (Calf Intestine Alkaline Phosphatase) is widely used. Conventionally, CIAP purified from calf small intestines is widely used, but in recent years, CIAP prepared by gene recombination has also become available commercially. However, the former is expensive to produce, and achieving stable quality is difficult. Further, in the case of the latter, production by expression in yeast or the like is used to reduce the production costs, but excessive glycosylation may occur, meaning problems relating to the background and viscosity and the like frequently occur. Further, although CIAP exhibits high activity, it suffers from poor stability, particularly thermal stability, and therefore maintaining the activity for long periods is difficult, and use of CIAP in gene-related applications which require heating is impossible. Moreover, because the activity decreases when diluted, in actual applications where the CIAP is used for long periods at low concentration, this property of high activity cannot be adequately realized.
BAP (Bacterial Alkaline Phosphatase) exhibits high stability, but because the activity is low, specifically only a few percent of the activity of CIAP, it has hardly been used at all for protein detection.
On the other hand, another method is known which, instead of using a labeled secondary antibody, uses an enzyme-labeled protein G prepared by binding an enzyme such as an alkaline phosphatase and protein G by chemical reaction. Protein G is a protein derived from the cell walls of streptococcal bacteria, and has a property of binding to the IgG of almost all mammals. By using this type of enzyme-labeled protein G, binding is possible with the primary antibodies of many immunity types, and even when a plurality of target materials (antigens) are to be detected, separate antibodies for binding specifically to each target material need not be prepared, thus offering excellent versatility.
However, methods using an enzyme-labeled protein G that has been labeled with an enzyme such as alkaline phosphatase have suffered from problems of low detection sensitivity. Further, thermal stability is also low, and loss of activity during handling may sometimes occur.
Non-Patent Document 1 discloses the preparation of a fusion protein in which a C1 domain of protein G (SpG) is bound to the N-terminal of Vargula hilgendorfii luciferase, but the fusion protein had no antibody binding ability, and a protein having a linker GGGGS inserted between the two moieties exhibited similar results.
Non-Patent Document 2 discloses the gene sequence, amino acid sequence, structure, and the function of each domain for protein G.
Non-Patent Document 3 discloses double mutants in which the amino acid residues at specific locations of BAP have been substituted, such as D153G/D330N and D153H/D330N, and examines the activity, stability, optimum pH, substrates, and metal ion affinity and activity of these mutants.
Patent Document 1 discloses mutations in which the amino acid residues at specific locations of BAP have been substituted, including mutants D153G and K328R, and the double mutant V99A/K328R, and discloses applications such as sandwich ELISA and competitive methods.
Patent Document 2 discloses mutations at position 329, position 330, and positions 153/328 of BAP, and discloses the preparation of a fusion protein with an antigen and the conducting of competitive ELISA.
Patent Document 3 discloses mutants of BAP such as K328R, and describes chemical binding to an antibody and ELISA.